title: Establishment and characterization of the reversibly immortalized mouse fetal heart progenitors.
abstract: OBJECTIVE
  Progenitor cell-based cardiomyocyte regeneration holds great promise of repairing an injured heart. Although cardiomyogenic differentiation has been reported for a variety of progenitor cell types, the biological factors that regulate effective cardiomyogenesis remain largely undefined. Primary cardiomyogenic progenitors (CPs) have a limited life span in culture, hampering the CPs' in vitro and in vivo studies. The objective of this study is to investigate if primary CPs isolated from fetal mouse heart can be reversibly immortalized with SV40 large T and maintain long-term cell proliferation without compromising cardiomyogenic differentiation potential.
  
  
  METHODS
  Primary cardiomyocytes were isolated from mouse E15.5 fetal heart, and immortalized retrovirally with the expression of SV40 large T antigen flanked with loxP sites. Expression of cardiomyogenic markers were determined by quantitative RT-PCR and immunofluorescence staining. The immortalization phenotype was reversed by using an adenovirus-mediated expression of the Cre reconbinase. Cardiomyogenic differentiation induced by retinoids or dexamethasone was assessed by an α-myosin heavy chain (MyHC) promoter-driven reporter.
  
  
  RESULTS
  We demonstrate that the CPs derived from mouse E15.5 fetal heart can be efficiently immortalized by SV40 T antigen. The conditionally immortalized CPs (iCP15 clones) exhibit an increased proliferative activity and are able to maintain long-term proliferation, which can be reversed by Cre recombinase. The iCP15 cells express cardiomyogenic markers and retain differentiation potential as they can undergo terminal differentiate into cardiomyctes under appropriate differentiation conditions although the iCP15 clones represent a large repertoire of CPs at various differentiation stages. The removal of SV40 large T increases the iCPs' differentiation potential. Thus, the iCPs not only maintain long-term cell proliferative activity but also retain cardiomyogenic differentiation potential.
  
  
  CONCLUSIONS
  Our results suggest that the reported reversible SV40 T antigen-mediated immortalization represents an efficient approach for establishing long-term culture of primary cardiomyogenic progenitors for basic and translational research.
authors.affiliation: Stem Cell Biology and Therapy Laboratory, the Key Laboratory of Pediatrics Designated by Chinese Ministry of Education and Chongqing Bureau of Education, and the Children's Hospital of Chongqing Medical University, Chongqing 400014, China.
authors.forename: Mi
authors.initials: M
authors.lastname: Li
authors.forename: Yuan
authors.initials: Y
authors.lastname: Chen
authors.forename: Yang
authors.initials: Y
authors.lastname: Bi
authors.forename: Wei
authors.initials: W
authors.lastname: Jiang
authors.forename: Qing
authors.initials: Q
authors.lastname: Luo
authors.forename: Yun
authors.initials: Y
authors.lastname: He
authors.forename: Yuxi
authors.initials: Y
authors.lastname: Su
authors.forename: Xing
authors.initials: X
authors.lastname: Liu
authors.forename: Jing
authors.initials: J
authors.lastname: Cui
authors.forename: Wenwen
authors.initials: W
authors.lastname: Zhang
authors.forename: Ruidong
authors.initials: R
authors.lastname: Li
authors.forename: Yuhan
authors.initials: Y
authors.lastname: Kong
authors.forename: Jiye
authors.initials: J
authors.lastname: Zhang
authors.forename: Jinhua
authors.initials: J
authors.lastname: Wang
authors.forename: Hongyu
authors.initials: H
authors.lastname: Zhang
authors.forename: Wei
authors.initials: W
authors.lastname: Shui
authors.forename: Ningning
authors.initials: N
authors.lastname: Wu
authors.forename: Jing
authors.initials: J
authors.lastname: Zhu
authors.forename: Jie
authors.initials: J
authors.lastname: Tian
authors.forename: Qi-Jian
authors.initials: QJ
authors.lastname: Yi
authors.forename: Hue H
authors.initials: HH
authors.lastname: Luu
authors.forename: Rex C
authors.initials: RC
authors.lastname: Haydon
authors.forename: Tong-Chuan
authors.initials: TC
authors.lastname: He
authors.forename: Gao-Hui
authors.initials: GH
authors.lastname: Zhu
mesh_terms: D000818:Animals; D002461:Cell Line, Transformed; D053595:Embryonic Stem Cells / Q000166:cytology*; D057809:HEK293 Cells; D006321:Heart / Q000196:embryology*; D006801:Humans; D051379:Mice; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction
keywords: Cardiomyogenic progenitors; cardiomyogenesis; cardiomyogenic differentiation; cardiovascular disorders; heart progenitor cells; immortalization